As part of our larger effort to understand unique aspects of HIV-1 biology that might be exploited in novel therapeutic approaches, we here focus on the critical role of chromatin in the transcriptional regulation of HIV-1. Chromatin has been recognized only recently as an important modulator of transcriptional regulatory mechanisms. Our most recent published studies indicate that nucleosomes are formed at specific and reproducible positions in the promoter of integrated HIV-1 in chronically infected cell lines. These nucleosomes delineate two regions devoid of nucleosomes located in the u3 region and immediately downstream of the 5' LTR. This nucleosomal organization is specifically disrupted inclose temporal association with transcriptional activation of the latent virus after TNF-alpha treatment. This finding, along with the observation that trapoxin and trichostatin A, two new specific inhibitors of histone deacetylase cause HIV-1 transcriptional activation, suggests that chromatin plays a repressive role in HIV-1 expression. We here propose several lines of investigation to clarify the role of chromatin in the regulation of HIV-1 transcription, with special attention on the mechanisms underlying latency and reactivation. We will determine the chromatin organization of HIV-1 by mapping 5'LTR nucleosome positions in several new cell lines carrying HIV-1 integrated at different sites; in lymphoid cell lines carrying the HIV-1 LTR stably maintained as an episome; and in vitro after reconstitution of the HIV-1LTR into chromatin with purified components. The effect of Tat, TNF-alpha and specific transcription factors on chromatin organization and on the functional activity of the promoter will be examined and a structure/function correlation will be established. To determine the mechanism of nucleosomal disruption during transcriptional activation, we will perform immunoprecipitation experiments using antisera specific for each of the histones and for acetylated vs unacetylated histones. The functional role of the nucleosome-free region located downstream of the 5'LTR will be determined by examining the biological phenotype of HIV-1 viruses containing mutations in several binding sites for transcription factors that have been identified in this region. It is expected that these studies will provide fundamental new insights into the process of HIV-1 transcriptional regulation and define novel targets for therapy aimed at interfering with HIV-1 replication by maintaining cells in the latent state and inhibiting reactivation from latency.